A mass spectrometry (MS) system in general includes an ion source for ionizing components of a sample under investigation, a mass analyzer for separating the ions based on their differing mass-to-charge ratios (or m/z ratios, or more simply “masses”), an ion detector for counting the separated ions, and electronics for processing output signals from the ion detector as needed to produce a user-interpretable mass spectrum. Typically, the mass spectrum is a series of peaks indicative of the relative abundances of detected ions as a function of their m/z ratios. The mass spectrum may be utilized to determine the molecular structures of components of the sample, thereby enabling the sample to be qualitatively and quantitatively characterized. One popular type of MS is the time-of-flight mass spectrometer (TOF MS). A TOF MS utilizes a high-resolution mass analyzer (TOF analyzer). Ions may be transported from the ion source into the TOF entrance region through a series of ion guides, ion optics, and various types of ion processing devices. The TOF analyzer includes an ion accelerator that injects ions in packets (or pulses) into an electric field-free flight tube. In the flight tube, ions of differing masses travel at different velocities and thus separate (spread out) according to their differing masses, enabling mass resolution based on time-of-flight.
Ion mobility spectrometry (IMS) is a gas-phase ion separation technique in which ions produced from a sample in an ion source are separated based on their differing mobilities through a drift cell of known length that is filled with an inert gas of known composition and maintained at a known gas pressure and temperature. In low-electric field drift-type IM, the ions are urged forward through the drift cell under the influence of a relatively weak, uniform DC voltage gradient. The mobility of the ions depends largely on their collision cross-sections (CCSs) and charge states (e.g., +1, +2, or +3), and to a much lesser extent their m/z ratios. Thus, ion separation by IM is largely orthogonal to ion separation by MS. From the drift cell the ions ultimately arrive at an ion detector, and the output signals from the ion detector are processed to generate peak information useful for distinguishing among the different analyte ion species detected.
An IMS system may be coupled online with a mass analyzer, which often is a TOF analyzer. In the combined IM-MS system, ions are separated by mobility prior to being transmitted into the mass analyzer where they are then mass-resolved. Due to the significant degree of orthogonality between IM-based separation and MS-based separation, performing the two separation techniques in tandem is particularly useful in the analysis of complex chemical mixtures, including biopolymers such as polynucleotides, proteins, carbohydrates and the like. For example, the added dimension provided by the IM separation may help to separate ions that are different from each other (e.g., in shape) but present overlapping mass peaks. On the other hand, the added dimension provided by the MS separation may help to separate ions that have different masses but similar CCSs. This hybrid IM-MS separation technique may be further enhanced by coupling it with liquid chromatography (LC) or gas chromatography (GC) techniques.
An IM-MS system is capable of acquiring multi-dimensional (IM-MS) data from a sample, characterized by acquisition time (i.e., chromatographic time or retention time), ion abundance (e.g., ion signal intensity), ion drift time through the IM drift cell, and m/z ratio as sorted by the MS. The IM-MS data may be quite complex and contain a very large number of data points, and hence may be difficult to evaluate by a researcher or user of the IM-MS system. This is particularly the case when the IM-MS data are acquired from a sample containing several species of high-molecular weight (MW) (bio)polymers such as proteins, peptides, and the like.
An “all-ions” IM-MS data acquisition may be performed in which all ions produced from a sample undergo separation in the IM drift cell and subsequently in the mass analyzer, without any active filtering of specific ions being undertaken prior to the acquisition. An all-ions IM-MS data acquisition may thus be characterized as a data-independent acquisition (DIA) experiment that produces a comprehensive IM-MS analysis of the sample. However, the resulting IM-MS data is of the type noted above that is complex and difficult to evaluate.
In a data-dependent acquisition (DDA) experiment, an all-ions IM-MS data acquisition may serve as the basis for finding and selecting one or more individual analyte ions of interest for further, second-stage analysis. Such selected ions may be further analyzed by operating a mass filter to sequentially isolate the selected ions from all other ions received in the mass filter, and sequentially acquiring spectral data (particularly fragment spectra) from the isolated ions. In particular, the isolated ions may be subjected to an MS/MS analysis in which, after isolation by the mass filter, the isolated ions are fragmented into fragment ions, and the fragment ions are then transmitted through a final mass analyzer to produce fragment spectra.
To aid in the selection of candidate ions for MS/MS analysis, software programs have been developed that provide graphical user interfaces (GUIs) configured to display IM-MS data in formats helpful to the user, but such GUIs generally provide less than complete solutions for aiding in the evaluation of complex IM-MS data. Other software programs, sometimes referred to as feature finders or feature extraction software, have also been developed that execute algorithms configured to find and select analyte ions of interest from a set of all-ions IM-MS data in an automated or semi-automated manner. Such computer-executed algorithms may identify candidate ions of interest based on a variety of data-dependent criteria such as minimum signal intensity, charge state, isotope pattern, and specific m/z values provided on an inclusion list or an exclusion list. However, a complex data set often includes a significant amount of ion signal interference or background chemical noise, making the process of finding and selecting analyte ions of interest difficult even when assisted by computer-executed algorithms. Thus, some ions that would be of actual interest may be overlooked due to signal interference and/or due to being present in low abundance (and thus being obscured by noise or not meeting a prescribed signal intensity threshold).
Therefore, there is a need for providing a DDA-type method that improves the process of evaluating potentially complex spectra, such as may for example be generated from a comprehensive IM-MS analysis such as an all-ions IM-MS data acquisition.